Phenylalanine hydroxylase (PheH) catalyzes the hydroxylation of dietary phenylalanine to tyrosine. Lack of a functional PheH results in the metabolic disease phenylketonuria (PKU). PheH is one of three aromatic amino acid hydroxylases; the other two are tyrosine hydroxylase and tryptophan hydroxylase. Regulation of PheH is tightly controlled so that only excess phenylalanine is consumed. The enzyme is regulated by activation by phenylalanine, inhibition by tetrahydrobiopterin (BH4), and phosphorylation at Ser16. The goal of this proposal is to understand how these regulatory mechanisms work together to control phenylalanine consumption. I propose to do this by 1) determining how phenylalanine binding causes the activation of PheH and 2) determining how pterin inhibits the activation of PheH. The kinetic parameters for phenylalanine activation will be determined by stopped-flow fluorescence spectroscopy, using the wild-type PheH, phosphorylated PheH and ?24 PheH. The stoichiometry and the energetics of phenylalanine-binding to these enzymes will determined by isothermal calorimetry (ITC). The effect of two PKU mutants, I65T and R68S, on activation of PheH will be determined. The kinetic parameters of pterin binding (BH4, 6MPH4, and the air-stable 5-deaza-6-methyltetrahdropterin) will be determined by stopped-flow absorbance spectroscopy. These experiments will be repeated with the E286A and S23A mutants. ITC will used to determine the stoichiometry and energetics of pterin binding and to analyze binding of pterin in the active site in the two mutants. 5-Deaza-6-methyltetrahdropterin and BH4 will be used to determine the effects of pterin on activation of PheH. This proposal is innovative because of the novel mechanism of activation. These studies will provide significant impact on the understanding of the mechanism for the regulation of PheH and the roles of PKU-causing mutations.